The characterization of a gas-phase ion primary structure may rely on information that can be obtained from the dissociation thereof. In the context of proteomics, protein identification and characterization may be conducted via tandem mass spectrometry using one or more gas-phase ion dissociation techniques. A commonly used method for activating polypeptide or protein ions is collision-induced dissociation (CID), which involves energetic collisions between ions and inert neutral bath gas atoms or molecules.
CID may be implemented with commercially available tandem mass spectrometers subject to a variety of operating conditions. These conditions may range from the slow activation (ms to s) low-collision-energy regime (<100 eV), of an ion trapping instrument (e.g., electrodynamic ion trap or penning trap) to the fast activation (μs), high collision energy regime (keV), in a beam-type instrument (e.g., a sector or time-of-flight/time-of-flight instrument).
Although the large differences in energies and time-scales associated with the various CID conditions can give rise to significant differences in the relative contributions of the competing peptide ion dissociation channels, structurally informative amide bond cleavages are generally observed, giving rise to b- and y-type ions.
Other than collision with inert gas species, methods using collision with surfaces, termed as surface-induced dissociation have been developed and applied to peptide dissociation studies. Photo-dissociation techniques, including infrared multi-photon dissociation (IRMPD), blackbody infrared dissociation (BIRD) and single-photon UV-photo-dissociation also show utility in providing structural information, which in some cases can complement that derived from other dissociation methods.
In addition to the techniques described above, the informative dissociation of multiply charged peptide and protein cations arising from the capture of low energy electrons, a phenomenon referred to as electron capture dissociation (ECD) may be employed. In ECD, the N—Cα bonds of the peptide backbone are cleaved, giving rise to sequence informative c- and z-type complementary ions. Compared to CID, ECD exhibits less sequence dependence on the cleavage sites and preservation of the post-translational modifications (PTMs), allowing characterization of modified protein ions.
The ion/ion reaction analogue to ECD is electron transfer dissociation (ETD), where the electron is transferred (ET) from an anion to a multiply-charged peptide or protein cation. ETD has been implemented on electrodynamic ion traps and it has been suggested that the dissociation due to electron transfer is similar to that observed in ECD. Accompanying dissociation products caused by electron transfer during the positive and negative ion encounters are contributions of varying abundance from electron transfer without subsequent dissociation (ET no D) and proton transfer (PT). The degree of competition from PT appears to be related to the characteristics of the anion reagent and the cation itself. Proton transfer can be minimized by the selection of the anionic reagent but no reagent appears to show exclusive electron transfer. The relative abundance of ET no D products has been observed to be strongly affected by the charge state of a peptide ion (e.g., much higher ET no D for lower charge states), as well as the identity of the protonated sites.
Elevated bath gas temperatures have been used in electron transfer ion/ion reactions. However, an improvement of ETD yields relative to room temperature ETD experiments is not consistently observed.
FIG. 1 shows four ways to effect ion/ion electron transfer dissociation reactions within a linear ion trap (LIT), where both polarity ions can be produced and injected into the LIT in an axial direction. One method involves the storage of neither ion polarity and relies on reactions taking place between the ions of opposite polarity as they are continuously admitted into the LIT (Method I). The likelihood for ion/ion reactions in this mode is expected to be the lowest of the four approaches because the relative velocities of the ions are the highest. Methods II and III involve storing one ion polarity while ions of the other polarity are continuously admitted into the LIT. Method IV employs mutual storage of oppositely charged ions, which is expected to provide the lowest relative velocities of the four approaches. The latter method requires the application of radio frequency (RF) voltages to the containment lenses of the LIT or the application of unbalanced RF to the quadrupole array.
Methods II and III of transmission mode electron transfer ion/ion reactions are taught in U.S. patent application Ser. No. 11/998,306 filed on Nov. 29, 2007, and which is incorporated herein by reference. (In the reference application, the corresponding methods are Methods I and II.)
Ion trap collisional activation of the ET no D products in a 3-D and a linear ion trap (LIT) appears to result in the formation of c- and z-type of ions, which tend to be complementary to those formed directly from ETD. This method has been demonstrated to be effective both in improving ETD yields and in increasing the extent of structural information from ETD with minimal contribution from dissociation of PT products that may also be present.
Beam-type post-ion/ion reaction collisional activation has been performed on a triple quadrupole/linear ion trap (LIT) system, where the ion/ion products, including the surviving precursor ions, are accelerated axially from a second LIT, in the presence of roughly 1 mTorr of nitrogen where the ion/ion reaction occurs in an adjacent first LIT for subsequent mass analysis. By choosing an appropriate acceleration potential, a significant increase in ETD yields can be achieved, although the contribution from the CID of precursor ions and other product ions does not appear to have been eliminated.
These methods for converting ET no D products into ETD products are based on a mutual trapping ion/ion reaction configuration, such as shown in method IV, where positive and negative ions are stored simultaneously in an overlapping space.